Probe, probe set, probe-immobilized carrier, and genetic testing method

ABSTRACT

A nucleic acid probe for classification of pathogenic bacterial species is capable of collectively detecting bacterial strains of the same species and differentially detecting them from other bacterial species. Any one of the base sequences of SEQ ID NOS. 73 to 75 and complementary or modified sequences thereof or a combination of at least two of them is used for detecting the gene of an infectious disease pathogenic bacterium.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to The present invention relates to aprobe and a probe set for detecting a gene of infectious diseasepathogenic bacterium, Flavobacterium (genus), which are useful fordetection and identification of the causative organism of an infectiousdisease, a probe-immobilized carrier on which the probe or the probe setis immobilized, a genetic testing method using the probe-immobilizedcarrier, and a genetic testing kit to be used for the method.

2. Description of the Related Art

Heretofore, reagents for and methods of quickly and accurately detectingthe causative organisms of infectious diseases in analytes have beenproposed. For instance, Japanese Patent Application Laid-Open No.H08-089254 discloses oligonucleotides having specific base sequences,which can be respectively used as probes and primers for detectingpathogenic bacteria of candidiasis and aspergillosis, and a method ofdetecting target bacteria using such oligonucleotides. In addition, thesame patent document also discloses a set of primers used forconcurrently amplifying a plurality of target bacteria by PCR. In otherwords, those primers are used for the PCR amplification of nucleic acidfragments from fungi, which serve as a plurality of targets, in ananalyte. Target fungal species in the analyte can be identified bydetecting the presence of a specific part of the sequence by ahybridization assay using probes specific to the respective fungi andthe nucleic acid fragments amplified by the respective primers.

On the other hand, the method to use probe array in which probes havingsequences complementary to the respective base sequences are arranged atintervals on a solid support is known as a method capable ofsimultaneously detecting a plurality of oligonucleotides havingdifferent base sequences (Japanese Patent Application Laid-Open No.2004-313181).

SUMMARY OF THE INVENTION

However, it is no easy task to design a probe for specifically detectinga gene of an infectious disease pathogenic bacterium in a sample. Thatis, as well as the target gene, the sample may further contain genes ofother infectious disease pathogenic bacteria. Thus, it is no easy taskto design the probe that specifically detects the gene of the infectiousdisease pathogenic bacterium while suppressing the cross contaminationwhich is the influence of the presence of the genes of other infectiousdisease pathogenic bacteria. Under such circumstances, the inventors ofthe present invention have studied for obtaining a probe which allowsaccurate detection of a gene of an infectious disease pathogenicbacterium as mentioned hereinbelow while maintaining the crosscontamination level low even when a sample in which genes of differentbacteria are present is used. As a result, the inventors of the presentinvention have finally found a plurality of probes capable of preciselydetecting the gene of the infectious disease pathogenic bacterium,Flavobacterium (genus).

A first object of the present invention is to provide a probe and aprobe set, which can precisely identify a gene of a target bacteriumfrom an analyte in which various bacteria are concurrently present.Another object of the present invention is to provide aprobe-immobilized carrier which can be used for precisely identifying atarget bacterium from an analyte in which various bacteria areconcurrently present. Still another object of the present invention isto provide a genetic testing method for detecting a target bacterium,which can quickly and precisely detect the target bacterium from variousbacteria in an analyte when they are present therein, and a kit for sucha method.

The probe for detecting a gene of infectious disease pathogenicbacterium, Flavobacterium (genus), of the present invention has any oneof the following base sequences (1) to (4):

(1) GGAAGTAACTAGAATATGTAGTGTAGCGGTG (SEQ ID NO. 73) or a complementarysequence thereof;

(2) GCCAGTGCAAACTGTGAGGAAGGT (SEQ ID NO. 74) or a complementary sequencethereof;

(3) GGGTAGGGGTCCTGAGAGGGAGATC (SEQ ID NO. 75) or a complementarysequence thereof; and

(4) a modified sequence prepared such that any one of the sequences ofSEQ ID NOS. 73 to 75 and the complementary sequences thereof issubjected to base deletion, substitution, or addition as far as themodified sequence retains a function as the probe.

In addition, the probe set for detecting a gene of infectious diseasepathogenic bacterium, Flavobacterium (genus), of the present inventionincludes at least two probes selected from the following items (A) to(L):

(A) a probe having a base sequence represented byGGAAGTAACTAGAATATGTAGTGTAGCGGTG (SEQ ID NO. 73);

(B) a probe having a base sequence represented byGCCAGTGCAAACTGTGAGGAAGGT (SEQ ID NO. 74);

(C) a probe having a base sequence represented byGGGTAGGGGTCCTGAGAGGGAGATC (SEQ ID NO. 75);

(D) a probe having a complementary sequence of the base sequencerepresented by SEQ ID NO. 73;

(E) a probe having a complementary sequence of the base sequencerepresented by SEQ ID NO. 74;

(F) a probe having a complementary sequence of the base sequencerepresented by SEQ ID NO. 75;

(G) a probe having a modified sequence obtained by base deletion,substitution, or addition on the base sequence represented by SEQ ID NO.73 as far as it retains the function of a probe for detecting the geneof Flavobacterium (genus);

(H) a probe having a modified sequence obtained by base deletion,substitution, or addition on the base sequence represented by SEQ ID NO.74 as far as it retains the function of a probe for detecting the geneof Flavobacterium (genus);

(I) a probe having a modified sequence obtained by base deletion,substitution, or addition on the base sequence represented by SEQ ID NO.75 as far as it retains the function of a probe for detecting the geneof Flavobacterium (genus);

(J) a probe having a modified sequence obtained by base deletion,substitution, or addition on the complementary sequence of the basesequence represented by SEQ ID NO. 73 as far as it retains the functionof a probe for detecting the gene of Flavobacterium (genus);

(K) a probe having a modified sequence obtained by base deletion,substitution, or addition on the complementary sequence of the basesequence represented by SEQ ID NO. 74 as far as it retains the functionof a probe for detecting the gene of Flavobacterium (genus); and

(L) a probe having a modified sequence obtained by base deletion,substitution, or addition on the complementary sequence of the basesequence represented by SEQ ID NO. 75 as far as it retains the functionof a probe for detecting the gene of Flavobacterium (genus).

The characteristic feature of the probe-immobilized carrier of thepresent invention is that at least one of the above-mentioned probes (A)to (L) is immobilized on a solid-phase carrier, and when a plurality ofprobes are employed, the respective probes are arranged at intervals.

The method of detecting a gene of an infectious disease pathogenicbacterium, Flavobacterium (genus), in an analyte by using aprobe-immobilized carrier of the present invention includes the stepsof:

(i) reacting the analyte with the probe-immobilized carrier having theabove-mentioned constitution; and

(ii) detecting the presence or absence of a reaction of the probe on theprobe-immobilized carrier with a nucleic acid in the analyte, ordetecting the strength of a hybridization reaction of the probe on theprobe-immobilized carrier with a nucleic acid in the analyte.

The characteristic feature of the kit for detecting an infectiousdisease pathogenic bacterium, Flavobacterium (genus), of the presentinvention is to include at least one of the above-mentioned probes (A)to (L), and a reagent for detecting a reaction between the probe and atarget nucleic acid.

According to the present invention, when an analyte is infected with theabove-mentioned causative bacterium, the bacterium can be more quicklyand precisely identified from the analyte even if the analyte issimultaneously and complexly infected with other bacteria in addition tothe above-mentioned bacterium. In particular, Flavobacterium (genus) canbe detected while precisely distinguishing it from Escherichia coliwhich may otherwise cause cross contamination.

Further features of the present invention will become apparent from thefollowing description of exemplary embodiments with reference to theattached drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram illustrating a 1st PCR protocol.

FIG. 2 is a diagram illustrating a 2nd PCR protocol.

DESCRIPTION OF THE EMBODIMENTS

The inventors of the present invention have obtained almost all ofbacteria (represented by (1) to (80) below), which have been known assepticemia pathogenic bacteria so far, from the respective depositoryinstitutions and identified the 16S rRNA gene sequences of all thebacteria.

Subsequently, while making a comparison of all the identified sequences,probe sequences for Flavobacterium (genus) were investigated in detailand the probes of the present invention, which can identifyFlavobacterium (genus), have finally been found out.

(1) Staphylococcus aureus (ATCC12600) (2) Staphylococcus epidermidis(ATCC14990) (3) Escherichia coli (ATCC11775) (4) Klebsiella pneumoniae(ATCC13883) (5) Pseudomonas aeruginosa (ATCC10145) (6) Serratiamarcescens (ATCC13380) (7) Streptococcus pneumoniae (ATCC33400) (8)Haemophilus influenzae (ATCC33391) (9) Enterobacter cloacae (ATCC13047)(10) Enterococcus faecalis (ATCC19433) (11) Staphylococcus haemolyticus(ATCC29970) (12) Staphylococcus hominis (ATCC27844) (13) Staphylococcussaprophyticus (ATCC15305) (14) Streptococcus agalactiae (ATCC13813) (15)Streptococcus mutans (ATCC25175) (16) Streptococcus pyogenes (ATCC12344)(17) Streptococcus sanguinis (ATCC10556) (18) Enterococcus avium(JCM8722) (19) Enterococcus faecium (ATCC19434) (20) Pseudomonasfluorescens (ATCC13525) (21) Pseudomonas putida (ATCC12633) (22)Burkholderia cepacia (JCM5964) (23) Stenotrophomonas maltophilia(ATCC13637) (24) Acinetobacter baumannii (ATCC19606) (25) Acinetobactercalcoaceticus (ATCC23055) (26) Achromobacter xylosoxidans (ATCC27061)(27) Vibrio vulnificus (ATCC27562) (28) Salmonella choleraesuis(JCM1651) (29) Citrobacter freundii (ATCC8090) (30) Klebsiella oxytoca(ATCC13182) (31) Enterobacter aerogenes (ATCC13048) (32) Hafnia alvei(ATCC13337) (33) Serratia liquefaciens (ATCC27592) (34) Proteusmirabilis (ATCC29906) (35) Proteus vulgaris (ATCC33420) (36) Morganellamorganii (ATCC25830) (37) Providencia rettgeri (JCM1675) (38) Aeromonashydrophila (JCM1027) (39) Aeromonas sobria (ATCC43979) (40) Gardnerellavaginalis (ATCC14018) (41) Corynebacterium diphtheriae (ATCC2701) (42)Legionella pneumophila (ATCC33152) (43) Bacillus cereus (ATCC14579) (44)Bacillus subtilis (ATCC6051) (45) Mycobacterium kansasii (ATCC12478)(46) Mycobacterium intracellulare (ATCC13950) (47) Mycobacteriumchelonae (ATCC35752) (48) Nocardia asteroids (ATCC19247) (49)Bacteroides fragilis (JCM11019) (50) Bacteroides thetaiotaomicron(JCM5827) (51) Clostridium difficile (ATCC51695) (52) Clostridiumperfringens (JCM1290) (53) Eggerthella lenta (JCM10763) (54)Fusobacterium necrophorum (JCM3718) (55) Fusobacterium nucleatum(ATCC25586) (56) Lactobacillus acidophilus (ATCC4356) (57) Anaerococcusprevotii (JCM6490) (58) Peptoniphilus asaccharolyticus (JCM8143) (59)Porphyromonas asaccharolytica (JCM6326) (60) Porphyromonas gingivalis(JCM8525) (61) Prevotella denticola (ATCC38184) (62) Propionibacteriumacnes (JCM6473) (63) Acinetobacter johnsonii (ATCC17909) (64)Acinetobacter junii (ATCC17908) (65) Aeromonas schubertii (ATCC43700)(66) Aeromonas veronii (ATCC35624) (67) Bacteroides distasonis(ATCC8503) (68) Bacteroides vulgatus (ATCC8482) (69) Campylobacter coli(ATCC33559) (70) Campylobacter hyointestinalis (ATCC35217) (71)Campylobacter jejuni (ATCC33560) (72) Flavobacterium aquatile(ATCC11947) (73) Flavobacterium mizutaii (ATCC33299) (74) Peptococcusniger (ATCC27731) (75) Propionibacterium avidum (ATCC25577) (76)Propionibacterium freudenreichii (ATCC6207) (77) Propionibacteriumgranulosum (ATCC25564) (78) Clostridium butyricum (ATCC13949) (79)Flavobacterium hydatis (NBRC14958) (80) Flavobacterium johnsoniae(NBRC14942)

The deposition numbers of the bacterial species obtained are shown inthe respective parentheses on the right side in the above. Bacterialspecies having deposition numbers beginning with “ATCC”, “JCM” and“NBRC” are available from American Type Culture Collection, JapanCollection of Microorganisms (RIKEN BioResource Center) and NationalBoard for Respiratory Care, respectively.

The present invention provides an oligonucleotide probe for identifyingan infectious disease pathogenic bacterium (hereinafter, simply referredto as a probe) and a probe set including a combination of two or moreprobes. The use of such a probe or a probe set allows the detection ofthe following bacterium which will cause inflammation by infection.

[Bacterial Name]

Flavobacterium (Genus)

That is, the probe of the present invention can detect the 16S rRNA genesequence among genes of the above-mentioned bacterium, having thefollowing sequences:

(A) a probe having a base sequence represented byGGAAGTAACTAGAATATGTAGTGTAGCGGTG (SEQ ID NO. 73);

(B) a probe having a base sequence represented byGCCAGTGCAAACTGTGAGGAAGGT (SEQ ID NO. 74);

(C) a probe having a base sequence represented byGGGTAGGGGTCCTGAGAGGGAGATC (SEQ ID NO. 75);

(D) a probe having a complementary sequence of the base sequencerepresented by SEQ ID NO. 73;

(E) a probe having a complementary sequence of the base sequencerepresented by SEQ ID NO. 74;

(F) a probe having a complementary sequence of the base sequencerepresented by SEQ ID NO. 75;

(G) a probe having a modified sequence obtained by base deletion,substitution, or addition on the base sequence represented by SEQ ID NO.73 as far as it retains the function of a probe for detecting the geneof Flavobacterium (genus);

(H) a probe having a modified sequence obtained by base deletion,substitution, or addition on the base sequence represented by SEQ ID NO.74 as far as it retains the function of a probe for detecting the geneof Flavobacterium (genus);

(I) a probe having a modified sequence obtained by base deletion,substitution, or addition on the base sequence represented by SEQ ID NO.75 as far as it retains the function of a probe for detecting the geneof Flavobacterium (genus);

(J) a probe having a modified sequence obtained by base deletion,substitution, or addition on the complementary sequence of the basesequence represented by SEQ ID NO. 73 as far as it retains the functionof a probe for detecting the gene of Flavobacterium (genus);

(K) a probe having a modified sequence obtained by base deletion,substitution, or addition on the complementary sequence of the basesequence represented by SEQ ID NO. 74 as far as it retains the functionof a probe for detecting the gene of Flavobacterium (genus); and

(L) a probe having a modified sequence obtained by base deletion,substitution, or addition on the complementary sequence of the basesequence represented by SEQ ID NO. 75 as far as it retains the functionof a probe for detecting the gene of Flavobacterium (genus).

The probe set can be formed using at least two of those probes.

The functions of those probes significantly depend on the specificity ofeach probe sequence corresponding to the target nucleic acid sequence ofinterest. The specificity of a probe sequence can be evaluated from thedegree of coincidence of bases with the target nucleic acid sequence andthe probe sequence. Further, when a plurality of probes constitute aprobe set, the variance of melting temperatures among the probes mayaffect the performance of the probe set.

For designing a probe sequence, a region showing a high specificity to aspecific bacterial species of interest regardless of any differences instrain is selected. The region contains three or more bases which arenot coincident with corresponding bases in the sequences of any otherbacterial species. The probe sequence is designed so that the meltingtemperature between the probe sequence and the corresponding sequence ofthe specific bacterial species of interest will differ by 10° C. or morefrom the melting temperatures between the probe sequence and thecorresponding sequences of any other bacterial species. Further, one ormore bases can be deleted or added so that the respective probesimmobilized on a single carrier may have melting temperatures within apredetermined range.

The inventors of the present invention found out by experiments that thehybridization intensity of a probe will not be significantly attenuatedif 80% or more of the base sequence is consecutively conserved. It cantherefore be concluded, from the finding, such that any sequencesmodified from the probe sequences disclosed in the specification willhave a sufficient probe function if 80% or more of the base sequence ofthe probe is consecutively conserved.

The above-mentioned modified sequences may include any variation as faras it does not impair the probe's function, or any variation as far asit hybridizes with a nucleic acid sequence of interest as a detectiontarget. Above all, it is desirable to include any variation as far as itcan hybridize with a nucleic acid sequence of interest as a detectiontarget under stringent conditions. Preferable hybridization conditionsconfining the variation include those represented in examples asdescribed below. Here, the term “detection target” used herein may beone included in a sample to be used in hybridization, which may be aunique base sequence to the infectious disease pathogenic bacterium, ormay be a complementary sequence to the unique sequence. Further, thevariation may be a modified sequence obtained by deletion, substitution,or addition of at least one base as far as it retains a function as theprobe.

Those probe sequences are only specific to the DNA sequence coding forthe 16S rRNA of the above-mentioned bacterium, so sufficienthybridization sensitivity to the sequence will be expected even understringent conditions. In addition, any of those probe sequences forms astable hybridized product through a hybridization reaction thereof witha target analyte even when the probe sequences are immobilized on acarrier, which is designed to produce an excellent result.

Further, a probe-immobilized carrier (e.g., DNA chip), on which theprobe for detecting the infectious disease pathogenic bacterium of thepresent invention, can be obtained by supplying the probe on apredetermined position on the carrier and immobilizing the probethereon. Various methods can be used for supplying the probe to thecarrier. Among them, for example, a method, which can be suitably used,is to keep a surface state capable of immobilizing the probe on thecarrier through a chemical bonding (e.g., covalent bonding) and a liquidcontaining the probe is then provided on a predetermined position by aninkjet method. Such a method allows the probe to be hardly detached fromthe carrier and exerts an additional effect of improving thesensitivity. In other words, when a stamping method conventionally usedand called the Stanford method is employed to make a DNA chip, theresultant DNA chip has a disadvantage such that the applied DNA tends tobe peeled off. Another one of the methods of forming DNA chips is tocarry out the arrangement of probes by the synthesis of DNA on thesurface of a carrier (e.g., DNA chip from Affymetrix Co., Ltd.). In sucha method of synthesizing probes on a carrier, it is difficult to makeequal the amount of synthesized DNA for each probe sequence. Thus, theamount of immobilized probe per immobilization area (spot) for eachprobe tends to vary considerably. Such variations in amounts of therespective immobilized probes may cause incorrect evaluation on theresults of the detection with those probes. Based on this fact, theprobe carrier of the present invention is preferably prepared using theabove-mentioned inkjet method. The inkjet method as described above hasan advantage such that the probe can be stably immobilized on thecarrier and hardly detaching from the carrier to efficiently provide aprobe carrier which can carry out detection with high sensitivity andhigh accuracy.

In addition, a probe set may include at least two selected from thegroup consisting of SEQ ID NOS. 73 to 75 as described above and thecomplementary sequences thereof and sequences obtained by base deletion,substitution, or addition on those sequences as far as they retain thefunction of a probe for detecting the gene of Flavobacterium (genus). Inthis case, the accuracy of detecting the Flavobacterium (genus) gene canbe further improved.

Hereinafter, preferred embodiments of the present invention will bedescribed in detail.

Test objects to be tested using probe carriers (e.g., DNA chips) inwhich the probes of the present invention are immobilized on carriersinclude those originated from humans and animals such as domesticanimals. For example, a test object is any of those which may containbacteria, including: any body fluids such as blood, cerebrospinal fluid,expectorated sputum, gastric juice, vaginal discharge, and oral mucosalfluid; and excretions such as urine and feces. All media, which can becontaminated with bacteria, can be also subjected to a test using a DNAchip. Such media include: food, drink water and water in the naturalenvironment such as hot spring water, which may cause food poisoning bycontamination; filters of air cleaners and the like; and so on. Animalsand plants, which should be quarantined in import/export, are also usedas analytes of interest.

When the sample as described above can be directly used in reaction withthe DNA chip, it is used as an analyte to react with the DNA chip andthe result of the reaction is then analyzed. Alternatively, when thesample cannot be directly reacted with the DNA chip, the sample wassubjected to extraction, purification, and other procedures forobtaining a target substance if required and then provided as an analyteto carry out a reaction with the DNA chip. For instance, when the samplecontains a target nucleic acid, an extract, which may be assumed tocontain such a target nucleic acid, is prepared from a sample, and thenwashed, diluted, or the like to obtain an analyte solution followed byreaction with the DNA chip. Further, as a target nucleic acid isincluded in an analyte obtained by carrying out various amplificationprocedures such as PCR amplification, the target nucleic acid may beamplified and then reacted with a DNA chip. Such analytes of amplifiednucleic acids include the following ones:

(a) An amplified analyte prepared by using a PCR-reaction primerdesigned for detecting 16S rRNA gene.

(b) An amplified analyte prepared by an additional PCR reaction or thelike from a PCR-amplified product.

(c) An analyte prepared by an amplification method other than PCR.

(d) An analyte labeled for visualization by any of various labelingmethods.

Further, a carrier used for preparing a probe-immobilized carrier, suchas a DNA chip, may be any of those that satisfy the property of carryingout a solid phase/liquid phase reaction of interest. Examples of thecarrier include: flat substrates such as a glass substrate, a plasticsubstrate, and a silicon wafer; a three-dimensional structure having anirregular surface; and a spherical body such as a bead, and rod-, cord-,and thread-shaped structures. The surface of the carrier may beprocessed such that a probe can be immobilized thereon. Especially, acarrier prepared by introducing a functional group to its surface toenable chemical reaction has a preferable form from the viewpoint ofreproducibility because the probe is stably bonded in the process ofhybridization reaction.

Various methods can be employed for the immobilization of probes. Anexample of such a method is to use a combination of a maleimide groupand a thiol (—SH) group. In this method, a thiol (—SH) group is bondedto the terminal of a probe, and a process is executed in advance to makethe carrier (solid) surface have a maleimide group. Accordingly, thethiol group of the probe supplied to the carrier surface reacts with themaleimide group on the carrier surface to form a covalent bond, wherebythe probe is immobilized.

Introduction of the maleimide group can utilize a process of firstlyallowing a reaction between a glass substrate and a aminosilane couplingagent and then introducing a maleimide group onto the glass substrate bya reaction of the amino group with an EMCS reagent(N-(6-maleimidocaproyloxy)succinimide, available from Dojindo).Introduction of the thiol group to a DNA can be carried out using5′-Thiol-Modifier C6 (available from Glen Research) when the DNA issynthesized by an automatic DNA synthesizer.

Instead of the above-described combination of a thiol group and amaleimide group, a combination of, e.g., an epoxy group (on the solidphase) and an amino group (nucleic acid probe terminal), can also beused as a combination of functional groups to be used forimmobilization. Surface treatments using various kinds of silanecoupling agents are also effective. A probe in which a functional groupwhich can react with a functional group introduced by a silane couplingagent is introduced is used. A method of applying a resin having afunctional group can also be used.

The detection of the gene of the infectious disease pathogenic bacteriumby using the probe-immobilized carrier of the present invention can becarried out by a genetic testing method including the steps of:

(i) reacting an analyte with a probe-immobilized carrier on which theprobe of the present invention is immobilized;

(ii) detecting the presence or absence of the reaction of a nucleic acidin the analyte with the probe on the probe-immobilized carrier, ordetecting the strength of the hybridization reaction of a nucleic acidin the analyte with the probe on the probe-immobilized carrier; and

(iii) specifying the probe having reacted with the nucleic acid in theanalyte when the reaction of the probe with the nucleic acid in theanalyte is detected and specifying the gene of the infectious diseasepathogenic bacterium in the analyte based on the nucleic acid sequenceof the probe.

The probe to be immobilized on the probe-immobilized carrier is at leastone of the above-mentioned items (A) to (L). On the carrier, probes fordetecting bacterial species other than Flavobacterium (genus) may beimmobilized as other probes, depending on the purpose of test. In thiscase, the other probes may be those capable of detecting the bacterialspecies other than Flavobacterium (genus) without causing crosscontamination and the use of such probes allows simultaneous detectionof a plurality of bacterial species with high accuracy.

Further, as described above, when the 16S rRNA gene sequence of aninfectious disease pathogenic bacterium in the analyte is amplified byPCR and provided as a sample to be reacted with a probe carrier, aprimer set for detecting the infectious disease pathogenic bacterium canbe used. The primer set suitably includes at least one selected fromoligonucleotides represented in the following items (1) to (21) and atleast one selected from oligonucleotides represented in the followingitems (22) to (28), more suitably includes all the oligonucleotidesrepresented in the following items (1) to (28):

(1) an oligonucleotide having a base sequence of 5′gcggcgtgcctaatacatgcaag 3′ (SEQ ID NO: 1);

(2) an oligonucleotide having a base sequence of 5′gcggcaggcctaacacatgcaag 3′ (SEQ ID NO: 2);

(3) an oligonucleotide having a base sequence of 5′gcggcaggcttaacacatgcaag 3′ (SEQ ID NO: 3);

(4) an oligonucleotide having a base sequence of 5′gcggtaggcctaacacatgcaag 3′ (SEQ ID NO: 4);

(5) an oligonucleotide having a base sequence of 5′gcggcgtgcttaacacatgcaag 3′ (SEQ ID NO: 5);

(6) an oligonucleotide having a base sequence of 5′gcgggatgccttacacatgcaag 3′ (SEQ ID NO: 6);

(7) an oligonucleotide having a base sequence of 5′gcggcatgccttacacatgcaag 3′ (SEQ ID NO: 7);

(8) an oligonucleotide having a base sequence of 5′gcggcatgcttaacacatgcaag 3′ (SEQ ID NO: 8);

(9) an oligonucleotide having a base sequence of 5′gcggcgtgcttaatacatgcaag 3′ (SEQ ID NO: 9);

(10) an oligonucleotide having a base sequence of 5′gcggcaggcctaatacatgcaag 3′ (SEQ ID NO: 10);

(11) an oligonucleotide having a base sequence of 5′gcgggatgctttacacatgcaag 3′ (SEQ ID NO: 11);

(12) an oligonucleotide having a base sequence of 5′gcggcgtgcctaacacatgcaag 3′ (SEQ ID NO: 12);

(13) an oligonucleotide having a base sequence of 5′gcggcgtgcataacacatgcaag 3′ (SEQ ID NO: 13);

(14) an oligonucleotide having a base sequence of 5′gcggcatgcctaacacatgcaag 3′ (SEQ ID NO: 14);

(15) an oligonucleotide having a base sequence of 5′gcggcgcgcctaacacatgcaag 3′ (SEQ ID NO: 15);

(16) an oligonucleotide having a base sequence of 5′gcggcgcgcttaacacatgcaag 3′ (SEQ ID NO: 16);

(17) an oligonucleotide having a base sequence of 5′gcgtcatgcctaacacatgcaag 3′ (SEQ ID NO: 17);

(18) an oligonucleotide having a base sequence of 5′gcgataggcttaacacatgcaag 3′ (SEQ ID NO: 18);

(19) an oligonucleotide having a base sequence of 5′gcgacaggcttaacacatgcaag 3′ (SEQ ID NO: 19);

(20) an oligonucleotide having a base sequence of 5′gctacaggcttaacacatgcaag 3′ (SEQ ID NO: 20);

(21) an oligonucleotide having a base sequence of 5′acagaatgcttaacacatgcaag 3′ (SEQ ID NO: 21);

(22) an oligonucleotide having a base sequence of 5′atccagccgcaccttccgatac 3′ (SEQ ID NO: 22);

(23) an oligonucleotide having a base sequence of 5′atccaaccgcaggttcccctac 3′ (SEQ ID NO: 23);

(24) an oligonucleotide having a base sequence of 5′atccagccgcaggttcccctac 3′ (SEQ ID NO: 24);

(25) an oligonucleotide having a base sequence of 5′atccagccgcaccttccggtac 3′ (SEQ ID NO: 25);

(26) an oligonucleotide having a base sequence of 5′atccagcgccaggttcccctag 3′ (SEQ ID NO: 26);

(27) an oligonucleotide having a base sequence of 5′atccagccgcaggttctcctac 3′ (SEQ ID NO: 27); and

(28) an oligonucleotide having a base sequence of 5′atccagccgcacgttcccgtac 3′ (SEQ ID NO: 28).

Among them, a primer designed for allowing the amplification ofFlavobacterium (genus) is a primer set of the following:

(3) an oligonucleotide having a base sequence of 5′gcggcaggcttaacacatgcaag 3′ (SEQ ID NO: 3); and

(25) an oligonucleotide having a base sequence of 5′atccagccgcaccttccggtac 3′ (SEQ ID NO: 25).

For detecting Flavobacterium (genus), at least such a primer may beincluded.

The utilities of the respective primers (1) to (28) for amplification ofFlavobacterium (genus) can be evaluated and confirmed by comparing eachsequence of SEQ ID NOs. 1 to 28 with a DNA sequence including the 16SrRNA coding region of Flavobacterium aquatile (ATCC 11947, SEQ ID NO.95).

A kit for detecting the infectious disease pathogenic bacterium can beconstructed using at least a probe as described above and a reagent fordetecting a reaction of the probe with a nucleic acid in an analyte. Theprobe in the kit can preferably be provided as a probe-immobilizedcarrier as described above. Further, the detection reagent may contain alabel to detect the reaction or a primer for carrying out amplificationas a pre-treatment.

EXAMPLES

Hereinafter, the present invention will be described in more detail withreference to examples using probes for detecting an infectious diseasepathogenic bacterium to detect Flavobacterium (genus).

Example 1

In this example, microorganism detection using 2-step PCR will bedescribed.

1. Preparation of Probe DNA

Nucleic acid sequences shown in Table 1 were designed as probes to beused for detection of Flavobacterium (genus). Specifically, thefollowing probe base sequences were selected from the genome part codingfor the 16s rRNA gene of Flavobacterium (genus). These probe basesequences were designed such that they could have an extremely highspecificity to the bacterium, and a sufficient hybridization sensitivitycould be expected without variance for the respective probe basesequences. The probe base sequences need not always completely matchwith those shown in Table 1. Probes having base lengths of 20 to 30which include the base sequences shown in Table 1 can also be used, inaddition to the probes having the base sequences shown in Table 1.However, it should be ensured that the other portion of the basesequence than the portion shown in Table 1 in such a probe has no effecton the detection accuracy.

TABLE 1 Name of microorganism Flavobacterium (genus) Probe No. SEQ IDNO. Sequence 111_G_FLA_01 73 5′ GGAAGTAACTAGAATATGTAGTGTAGCGGTG 3′111_G_FLA_02 74 5′ GCCAGTGCAAACTGTGAGGAAGGT 3′ 111_G_FLA_03 755′ GGGTAGGGGTCCTGAGAGGGAGATC 3′

For each probe having a base sequence shown in Table 1, a thiol groupwas introduced, as a functional group to immobilize the probe on a DNAchip, to the 5′ terminal of the nucleic acid after synthesis inaccordance with a conventional method. After introduction of thefunctional group, purification and freeze-drying were executed. Thefreeze-dried probes for internal standard were stored in a freezer at−30° C.

2. Preparation of PCR Primers

2-1. Preparation of PCR Primers for Analyte Amplification

As 16S rRNA gene (target gene) amplification PCR primers for pathogenicbacterium detection, nucleic acid sequences shown in Table 2 below weredesigned. Specifically, primer sets which specifically amplify thegenome parts coding the 16S rRNAs, i.e., primers for which the specificmelting points were made uniform as far as possible at the two endportions of the 16S rRNA coding region of a base length of 1,400 to1,700 were designed. In order to simultaneously amplify a plurality ofdifferent bacterial species listed in the following items (1) to (80),mutants, or a plurality of 16S rRNA genes on genomes, a plurality ofkinds of primers were designed. Note that a primer set is not limited tothe primer sets shown in Table 2 as far as the primer set is availablein common to amplify almost the entire lengths of the 16S rRNA genes ofthe pathogenic bacteria.

TABLE 2 Primer No SEQ ID NO. Sequence F01 1 5′ GCGGCGTGCCTAATACATGCAAG3′ F02 2 5′ GCGGCAGGCCTAACACATGCAAG 3′ F03 3 5′ GCGGCAGGCTTAACACATGCAAG3′ F04 4 5′ GCGGTAGGCCTAACACATGCAAG 3′ F05 5 5′ GCGGCGTGCTTAACACATGCAAG3′ F06 6 5′ GCGGGATGCCTTACACATGCAAG 3′ F07 7 5′ GCGGCATGCCTTACACATGCAAG3′ F08 8 5′ GCGGCATGCTTAACACATGCAAG 3′ F09 9 5′ GCGGCGTGCTTAATACATGCAAG3′ F10 10 5′ GCGGCAGGCCTAATACATGCAAG 3′ F11 115′ GCGGGATGCTTTACACATGCAAG 3′ F12 12 5′ GCGGCGTGCCTAACACATGCAAG 3′ F1313 5′ GCGGCGTGCATAACACATGCAAG 3′ F14 14 5′ GCGGCATGCCTAACACATGCAAG 3′F15 15 5′ GCGGCGCGCCTAACACATGCAAG 3′ F16 16 5′ GCGGCGCGCTTAACACATGCAAG3′ F17 17 5′ GCGTCATGCCTAACACATGCAAG 3′ F18 185′ GCGATAGGCTTAACACATGCAAG 3′ F19 19 5′ GCGACAGGCTTAACACATGCAAG 3′ F2020 5′ GCTACAGGCTTAACACATGCAAG 3′ F21 21 5′ ACAGAATGCTTAACACATGCAAG 3′R0l 22 5′ ATCCAGCCGCACCTTCCGATAC 3′ R02 23 5′ ATCCAACCGCAGGTTCCCCTAC 3′R03 24 5′ ATCCAGCCGCAGGTTCCCCTAC 3′ R04 25 5′ ATCCAGCCGCACCTTCCGGTAC 3′R05 26 5′ ATCCAGCGCCAGGTTCCCCTAG 3′ R06 27 5′ ATCCAGCCGCAGGTTCTCCTAC 3′R07 28 5′ ATCCAGCCGCACGTTCCCGTAC 3′

(1) Staphylococcus aureus

(2) Staphylococcus epidermidis

(3) Escherichia coli

(4) Klebsiella pneumoniae

(5) Pseudomonas aeruginosa

(6) Serratia marcescens

(7) Streptococcus pneumoniae

(8) Haemophilus influenzae

(9) Enterobacter cloacae

(10) Enterococcus faecalis

(11) Staphylococcus haemolyticus

(12) Staphylococcus hominis

(13) Staphylococcus saprophyticus

(14) Streptococcus agalactiae

(15) Streptococcus mutans

(16) Streptococcus pyogenes

(17) Streptococcus sanguinis

(18) Enterococcus avium

(19) Enterococcus faecium

(20) Pseudomonas fluorescent

(21) Pseudomonas putida

(22) Burkholderia cepacia

(23) Stenotrophomonas maltophilia

(24) Acinetobacter baumannii

(25) Acinetobacter calcoaceticus

(26) Achromobacter xylosoxidans

(27) Vibrio vulnificus

(28) Salmonella choleraesuis

(29) Citrobacter freundii

(30) Klebsiella oxytoca

(31) Enterobacter aerogenes

(32) Hafnia alvei

(33) Serratia liquefaciens

(34) Proteus mirabilis

(35) Proteus vulgaris

(36) Morganella morganii

(37) Providencia rettgeri

(38) Aeromonas hydrophila

(39) Aeromonas sobria

(40) Gardnerella vaginalis

(41) Corynebacterium diphtheriae

(42) Legionella pneumophila

(43) Bacillus cereus

(44) Bacillus subtilis

(45) Mycobacterium kansasii

(46) Mycobacterium intracellulare

(47) Mycobacterium chelonae

(48) Nocardia asteroides

(49) Bacteroides fragilis

(50) Bacteroides thetaiotaomicron

(51) Clostridium difficile

(52) Clostridium perfringens

(53) Eggerthella lenta

(54) Fusobacterium necrophorum

(55) Fusobacterium nucleatum

(56) Lactobacillus acidophilus

(57) Anaerococcus prevotii

(58) Peptoniphilus asaccharolyticus

(59) Porphyromonas asaccharolytica

(60) Porphyromonas gingivalis

(61) Prevotella denticola

(62) Propionibacterium acnes

(63) Acinetobacter johnsonii

(64) Acinetobacter junii

(65) Aeromonas schubertii

(66) Aeromonas veronii

(67) Bacteroides distasonis

(68) Bacteroides vulgatus

(69) Campylobacter coli

(70) Campylobacter hyointestinalis

(71) Campylobacter jejuni

(72) Flavobacterium aquatile

(73) Flavobacterium mizutaii

(74) Peptococcus niger

(75) Propionibacterium avidum

(76) Propionibacterium freudenreichii

(77) Propionibacterium granulosum

(78) Clostridium butyricum

(79) Flavobacterium hydatis

(80) Flavobacterium johnsoniae

The primers shown in Table 2 were purified by high performance liquidchromatography (HPLC) after synthesis. The twenty-one forward primersand the seven reverse primers were mixed and dissolved in a TE buffersolution such that each primer concentration had an ultimateconcentration of 10 pmol/μl.

2-2. Preparation of Labeling PCR Primers

In a manner similar to the above-mentioned analyte amplificationprimers, oligonucleotides having sequences as shown in Table 3 belowwere employed as primers for labeling.

TABLE 3 Primer No SEQ ID NO. Sequence Cy3R9-1 295′ TACCTTGTTACGACTTCACCCCA 3′ Cy3R9-2 30 5′ TACCTTGTTACGACTTCGTCCCA 3′Cy3R9-3 31 5′ TACCTTGTTACGACTTAGTCCCA 3′ Cy3R9-4 325′ TACCTTGTTACGACTTAGCCCCA 3′ Cy3R9-5 33 5′ TACCTTGTTACGACTTAGTCCTA 3′Cy3R9-6 34 5′ TACCTTGTTACGACTTAGCCCTA 3′

The primers shown in Table 3 were labeled with a fluorescent dye, Cy3.The primers were purified by high performance liquid chromatography(HPLC) after synthesis. The six labeled primers were mixed and dissolvedin a TE buffer solution such that each primer concentration had anultimate concentration of 10 pmol/μl.

3. Extraction of Genome DNA (Model Analyte) of Flavobacterium (Genus)

3-1. Microbial Culture & Genome DNA Extraction

First, three species of Flavobacterium (genus), Flavobacterium aquatile(ATCC 11947), Flavobacterium hydatis (NBRC 14958) and Flavobacteriumjohnsoniae (NBRC 14942), were cultured in accordance with theconventional method. This microbial culture medium was subjected to theextraction and purification of genome DNA by using a nucleic acidpurification kit (FastPrep FP100A FastDNA Kit, manufactured by FunakoshiCo., Ltd.).

3-2. Test of Collected Genome DNA

The collected genome DNA of the microorganism, Flavobacterium (genus),was subjected to agarose electrophoresis and 260/280-nm absorbancedetermination in accordance with the conventional method. Thus, thequality (the admixture amount of low molecular nucleic acid and thedegree of decomposition) and the collection amount were tested. In thisexample, about 10 μg of the genome DNA was collected. No degradation ofgenome DNA or contamination of rRNA was observed. The collected genomeDNA was dissolved in a TE buffer solution at an ultimate concentrationof 50 ng/μl and used in the following experiments.

4. Preparation of DNA Chip

4-1. Cleaning of Glass Substrate

A glass substrate (size: 25 mm×75 mm×1 mm, available from IiyamaPrecision Glass) made of synthetic quartz was placed in a heat- andalkali-resisting rack and dipped in a cleaning solution for ultrasoniccleaning, which was adjusted to have a predetermined concentration. Theglass substrate was kept dipped in the cleaning solution for a night andcleaned by ultrasonic cleaning for 20 min. The substrate was picked up,lightly rinsed with pure water, and cleaned by ultrasonic cleaning inultrapure water for 20 min. The substrate was dipped in a 1N aqueoussodium hydroxide solution heated to 80° C. for 10 min. Pure watercleaning and ultrapure water cleaning were executed again. A quartzglass substrate for a DNA chip was thus prepared.

4-2. Surface Treatment

A silane coupling agent KBM-603 (available from Shin-Etsu Silicone) wasdissolved in pure water at a concentration of 1% by weight (wt %) andstirred at room temperature for 2 hrs. The cleaned glass substrate wasdipped in the aqueous solution of the silane coupling agent and leftstand still at room temperature for 20 min. The glass substrate waspicked up. The surface thereof was lightly rinsed with pure water anddried by spraying nitrogen gas to both surfaces of the substrate. Thedried substrate was baked in an oven at 120° C. for 1 hr to complete thecoupling agent treatment, whereby an amino group was introduced to thesubstrate surface. Next, N-maleimidocaproyloxy succinimido (abbreviatedas EMCS hereinafter) was dissolved in a 1:1 (volume ratio) solventmixture of dimethyl sulfoxide and ethanol to obtain an ultimateconcentration of 0.3 mg/ml. As a result, an EMCS solution was prepared.Here, EMCS is N-(6-maleimidocaproyloxy)succinimido available fromDojindo.

The baked glass substrate was left stand and cooled and dipped in theprepared EMCS solution at room temperature for 2 hrs. By this treatment,the amino group introduced to the surface of the substrate by the silanecoupling agent reacted with the succinimide group in the EMCS tointroduce the maleimide group to the surface of the glass substrate. Theglass substrate picked up from the EMCS solution was cleaned by usingthe above-described solvent mixture in which the EMCS was dissolved. Theglass substrate was further cleaned by ethanol and dried in a nitrogengas atmosphere.

4-3. Probe DNA

The microorganism detection probe prepared in the stage 1 (Preparationof Probe DNA) of Example 1 was dissolved in pure water. The solution wasdispensed such that the ultimate concentration (at ink dissolution)became 10 μM. Then, the solution was freeze-dried to remove water.

4-4. DNA Discharge by BJ Printer and Bonding to Substrate

An aqueous solution containing 7.5-wt % glycerin, 7.5-wt % thiodiglycol,7.5-wt % urea, and 1.0-wt % Acetylenol EH (available from Kawaken FineChemicals) was prepared. Each of the three probes (Table 1) prepared inadvance was dissolved in the solvent mixture at a specificconcentration. An ink tank for an inkjet printer (trade name: BJF-850,available from Canon) is filled with the resultant DNA solution andattached to the printhead.

The inkjet printer used here was modified in advance to allow printingon a flat plate. When a printing pattern is input in accordance with apredetermined file creation method, an about 5-picoliter of a DNAsolution can be spotted at a pitch of about 120 μm.

The printing operation was executed for one glass substrate by using themodified inkjet printer to prepare an array. After confirming thatprinting was reliably executed, the glass substrate was left stand stillin a humidified chamber for 30 min to make the maleimide group on theglass substrate surface react with the thiol group at the nucleic acidprobe terminal.

4-5. Cleaning

After reaction for 30 min, the DNA solution remaining on the surface wascleaned by using a 10-mM phosphate buffer (pH 7.0) containing 100-mMNaCl, thereby obtaining a DNA chip in which single-stranded DNAs wereimmobilized on the glass substrate surface.

5. Amplification and Labeling of Analyte

5-1. Amplification of Analyte: 1st PCR

The amplification reaction (1st PCR) and the labeling reaction (2nd PCR)of a microbial gene to be provided as an analyte are shown in Table 4below.

TABLE 4 AmpliTaq Gold LD (5.0 U/μL) 0.5 μL (2.5 unit/tube) Primer mix<FR21x7> 2.0 μL Forward primer (x21 [0.625 μM/each]) (final 1.25 pmoleach/tube) Reverse primer (x07 [1.875 μM/each]) (final 3.75 pmoleach/tube) 10x PCR buffer 5.0 μL (final 1x conc.) MgCl₂ (25 mM) 8.0 μL(final 4.0 mM) dNTPmix (2.5 mM/each) 4.0 μL (final 200 μM each) Templatevariable H₂O up to 50 μL Total 50 μL

Amplification reaction of the reaction solution having theabove-mentioned composition was carried out using a commerciallyavailable thermal cycler in accordance with the protocol illustrated inFIG. 1. After the end of reaction, the primer was purified using apurification column (QIAquick PCR Purification Kit available fromQIAGEN). Subsequently, the quantitative assay of the amplified productwas carried out.

5-2. Labeling Reaction: 2nd PCR

Amplification reaction of the reaction solution having the compositionshown in Table 5 was carried out using a commercially available thermalcycler in accordance with the protocol illustrated in FIG. 2.

TABLE 5 Premix Taq (Ex Taq Version) 25 μL Cy3-labeled reverse primer mix0.83 μL Cy3R9 mix (x06[10 μM/each]) (final 8.3 pmol each/tube) Templatevariable (final 30 ng/tube) H₂O up to 50 μL Total 50 μL

After the end of reaction, the primer was purified using a purificationcolumn (QIAquick PCR Purification Kit available from QIAGEN) to obtain alabeled analyte.

6. Hybridization

Detection reaction was performed using the DNA chip prepared in thestage 4 (Preparation of DNA Chip) and the labeled analyte prepared inthe stage 5 (Amplification and Labeling of Analyte).

6-1. Blocking of DNA Chip

Bovine serum albumin (BSA, Fraction V: available from Sigma) wasdissolved in a 100-mM NaCl/10-mM phosphate buffer such that a 1 wt %solution was obtained. Then, the DNA chip prepared in the stage 4(Preparation of DNA Chip) was dipped in the solution at room temperaturefor 2 hrs to execute blocking. After the end of blocking, the chip wascleaned using a washing solution as described below, rinsed with purewater and hydro-extracted by a spin dryer.

The washing solution: 2×SSC solution (NaCl 300 mM, sodium citrate(trisodium citrate dihydrate, C₆H₅Na₃.2H₂O) 30 mM, pH 7.0) containing0.1-wt % sodium dodecyl sulfate (SDS)

6-2. Hybridization

The hydro-extracted DNA chip was placed in a hybridization apparatus(Hybridization Station available from Genomic Solutions Inc).Hybridization reaction was carried out in a hybridization solution underconditions as described below.

6-3. Hybridization Solution

6×SSPE/10% formamide/target (all 2nd PCR products)/0.05 wt % (6×SSPE:NaCl 900 mM, NaH₂PO₄H₂O 50 mM, EDTA 6 mM, pH, 7.4)

6-4. Hybridization Conditions

65° C. for 3 min, 55° C. for 4 hrs, washing with 2×SSC/0.1% SDS at 50°C., washing with 2×SSC/0.1% SDS at 20° C. (rinse with H₂O: manual), andspin dry.

7. Microorganism Genome Detection (Fluorometry)

The DNA chip after the end of hybridization reaction was subjected tofluorometry with a DNA chip fluorescent detector (GenePix 4000Bavailable from Axon). As a result, Flavobacterium (genus) was able to bedetected with a sufficient signal at a high reproducibility. The resultsof fluorometry are shown in Table 6 below.

TABLE 6 Fluorescence intensity Flavobacterium FlavobacteriumFlavobacterium aquatile hydatis johnsoniae Probe No. ATCC 11947 NBRC14958 NBRC 14942 111_G_FLA_01 9672.5 6582.3 1545.6 111_G_FLA_02 15516.28944.6 18464.5 111_G_FLA_03 2325.0 5415.4 5465.5

8. Hybridization with Other Bacterial Species

For proving the fact that the probe set shown in Table 1 can bespecifically hybridized only with Flavobacterium (genus), the results ofhybridization reaction with Escherichia coli (JCM 1649) are shown inTable 7 below.

TABLE 7 Fluorescence intensity Probe No. Escherichia coli (JCM 1649)111_G_FLA_01 50.3 111_G_FLA_02 149.7 111_G_FLA_03 50.0

9. Results

As is evident from the above description, a DNA chip was prepared suchthat a probe set, which was able to detect only Flavobacterium (genus)in a specific manner, was immobilized. Further, the use of such a DNAchip allowed the identification of an infectious disease pathogenicbacterium, so the problems of the DNA probe derived from a microorganismwas able to be solved. In other words, the oligonucleotide probe can bechemically produced in large amounts, while the purification orconcentration thereof can be controlled. In addition, for classificationof microbial species, a probe set capable of collectively detectingbacterial strains of the same genus and differentially detecting themfrom bacteria of other genera, was able to be provided.

Further, in addition to Escherichia coli as described above,hybridization reaction was carried out on each of nucleic acidsextracted from the bacteria represented in the above-mentioned items (1)to (80). The results thereof confirmed that no substantial reaction wasobserved with respect to each of those bacteria in a manner similar tothat of Escherichia coli, except of Flavobacterium (genus).

The bacteria represented in the above-mentioned items (1) to (80) arepathogenic bacteria for septicemia, and they cover almost all of thepathogenic bacteria ever detected in human blood. Therefore, by usingthe primer of the present embodiment, the nucleic acid of an infectiousdisease pathogenic bacterium in blood can be extracted and thensubjected to hybridization reaction with the probe of the presentinvention, whereby identification of Flavobacterium (genus) can beperformed with higher accuracy.

Further, according to the above-mentioned example, the presence of aninfectious disease pathogenic bacterium can be efficiently determinedwith high accuracy by completely detecting the 16S rRNA gene from thegene of the infectious disease pathogenic bacterium.

Example 2 Preparation of DNA Chip by which Various Bacterial Species canbe Simultaneously Determined

In a manner similar to the stage 1 (Preparation of Probe DNA) of Example1, probes having base sequences as shown in Table 8 below were prepared.

TABLE 8 Bacterial species (or SEQ ID genus) of interest Probe sequence(5′→3′) NO. Anaerococcus prevotii TCATCTTGAGGTATGGAAGGGAAAGTGG 35GTGTTAGGTGTCTGGAATAATCTGGGTG 36 ACCAAGTCTTGACATATTACGGCGG 37 Bacteroidesfragilis AAGGATTCCGGTAAAGGATGGGGATG 38 TGGAAACATGTCAGTGAGCAATCACC 39Bacteroides AAGAATTTCGGTTATCGATGGGGATGC 40 thetaiotaomicronAAGTTTTCCACGTGTGGAATTTTGTATGT 41 AAGGCAGCTACCTGGTGACAGGAT 42 Clostridiumdifficile AATATCAAAGGTGAGCCAGTACAGGATGGA 43 CCGTAGTAAGCTCTTGAAACTGGGAGAC44 TCCCAATGACATCTCCTTAATCGGAGAG 45 Clostridium perfringensAACCAAAGGAGCAATCCGCTATGAGAT 46 GAGCGTAGGCGGATGATTAAGTGGG 47CCCTTGCATTACTCTTAATCGAGGAAATC 48 Eggerthella lentaGGAAAGCCCAGACGGCAAGGGA 49 CCTCTCAAGCGGGATCTCTAATCCGA 50TGCCCCATGTTGCCAGCATTAGG 51 Fusobacterium necrophorumTTTTCGCATGGAGGAATCATGAAAGCTA 52 AGATGCGCCGGTGCCCTTTCG 53AGTCGGGAAGAAGTCAGTGACGGTAC 54 Peptoniphilus GAGTACGTGCGCAAGCATGAAACT 55asaccharolyticus Porphyromonas GAAGACTGCCCGCAAGGGTTGTAA 56asaccharolytica GTGTACTGGAGGTACGTGGAACGTG 57 GCATGAGGCTGAGAGGTCTCTTCC 58Porphyromonas gingivalis TTATAGCTGTAAGATAGGCATGCGTCCC 59AACGGGCGATACGAGTATTGCATTGA 60 ATATACCGTCAAGCTTCCACAGCGA 61 Enterococcusavium TTTGAAAGGCGCTTTTGCGTCACTG 62 CAAGGATGAGAGTAGAACGTTCATCCCTTG 63CAAGGATGAGAGTAGAATGTTCATCCCTTG 64 Providencia rettgeriCCTGGGAATGGCATCTAAGACTGGTCA 65 Acinetobacter(genus)GAGGAAGGCGTTGATGCTAATATCATCA 66 GAGCAAAGCAGGGGAACTTCGGTC 67GTTGGGGCCTTTGAGGCTTTAGTG 68 TGGGAGAGGATGGTAGAATTCCAGGT 69 Prevotelladenticola TCGATGACGGCATCAGATTCGAAGCA 70 AATGTAGGCGCCCAACGTCTGACT 71ATGTTGAGGTCCTTCGGGACTCCT 72 Flavobacterium(genus)GGAAGTAACTAGAATATGTAGTGTAGCGGTG 73 GCCAGTGCAAACTGTGAGGAAGGT 74GGGTAGGGGTCCTGAGAGGGAGATC 75 Aeromonas(genus) GAGTGCCTTCGGGAATCAGAACAC76 CTGCAAGCTAGCGATAGTGAGCGA 77 Bacteroides(genus)CGATGGATAGGGGTTCTGAGAGGAA 78 TGCGGCTCAACCGTAAAATTGCAGT 79TGTGGCTCAACCATAGAATTGCCGT 80 Peptococcus nigerGTACCTGTAAGAAAGACGGCCTTCGT 81 CTGCCGAGTGATGTAATGTCACTTTTC 82TCGGAGGTTTCAAGACCGTCGG 83 Clostridium(genus) ACCAAAGGAGCAATCCGCTATGAGATG84 ATCAAAGGTGAGCCAGTACAGGATGG 85 ATTAAAGGAGTAATCCGCTATGAGATGGACC 86Propionibacterium acnes GGGCTAATACCGGATAGGAGCTCCTG 87AAGCGTGAGTGACGGTAATGGGTAAA 88 ATCGCGTCGGAAGTGTAATCTTGGG 89Campylobacter(genus) TGGAGCAAATCTATAAAATATGTCCCAGT 90ACAGTGGAATCAGCGACTGGGG 91 Aeromonas hydrophilaGCCTAATACGTATCAACTGTGACGTTAC 92 GCCTAATACGTGTCAACTGTGACGTTAC 93Propionibacterium(genus) GCTTTCGATACGGGTTGACTTGAGGAA 94

Those probes are capable of specifically detecting certain bacterialspecies (or genera) shown in the left column in the table just as onespecific to Flavobacterium (genus) of Example 1.

Further, those probes are designed such that they have the same Tm valueas that of a target, the same reactivity with a non-target sequence, andthe like so that the nucleic acid of the bacterial species of interestcan be specifically detected under the same reaction conditions.

For the respective probes, probe solutions were prepared in a mannersimilar to the stage 4-3 of Example 1. Subsequently, the inkjet printerused in the stage 4-4 of Example 1 was employed to discharge each of theprobe solution on the same substrate to form a plurality of DNA chipshaving spots of the respective probes being arranged at a pitch of about120 μm.

One of the DNA chips was used for hybridization with the nucleic acidextracted from Flavobacterium (genus) in a manner similar to the stage 6of Example 1. As a result, the spot of the probe which specificallydetected Flavobacterium (genus) showed almost the equal fluorescenceintensity as that of Example 1. In contrast, the spots of other probesshowed extremely low fluorescence intensity.

Further, other prepared DNA chips were used for hybridization with thebacteria listed in Table 8 except of Flavobacterium (genus). As aresult, the spot of Flavobacterium (genus) showed extremely lowfluorescence intensity, while the spot of the probe for the bacterialspecies of interest showed extremely high fluorescence intensity.Therefore, the DNA chip prepared in the present example was confirmedthat it was able to simultaneously determine 16 bacterial species and 6genera listed in Table 8 in addition to Flavobacterium (genus). Bysimultaneously using probes for a target species and the correspondinggenus (for example, Propionibacterium (genus) and Propionibactriumacnes), highly accurate identification of the target species orsimultaneous identification of a plurality of target species of the samegenus can be performed.

Example 3

Using the DNA chip prepared in Example 2, detection was attempted when aplurality of bacterial species was present in an analyte.

A culture medium in which Flavobacterium aquatile as Flavobacterium(genus) and Eggerthella lenta were cultured was prepared and subjectedto the same treatment as that of Example 1 to react with the DNA chip.

As a result, only the spots of the probes having SEQ ID NOS. 49, 50, 51,73, 74, and 75 showed high fluorescence intensity, so the coexistence ofthose bacteria was able to be simultaneously confirmed.

The present invention is not limited to the above-mentioned embodimentsand various changes and modifications can be made within the spirit andscope of the present invention. Therefore to apprise the public of thescope of the present invention, the following claims are made.

This application claims the benefit of Japanese Patent Application No.2006-306005, filed Nov. 10, 2006, which is hereby incorporated byreference in its entirety.

1. A probe for detecting a gene of infectious disease pathogenicbacterium, Flavobacterium (genus), having any one of the following basesequences (1) to (4): (1) GGAAGTAACTAGAATATGTAGTGTAGCGGTG (SEQ ID NO.73) or a complementary sequence thereof; (2) GCCAGTGCAAACTGTGAGGAAGGT(SEQ ID NO. 74) or a complementary sequence thereof; (3)GGGTAGGGGTCCTGAGAGGGAGATC (SEQ ID NO. 75) or a complementary sequencethereof; and (4) a modified sequence prepared such that any one of thesequences of SEQ ID NOS. 73 to 75 and the complementary sequencesthereof is subjected to base deletion, substitution, or addition as faras the modified sequence retains a function as the probe.
 2. Aprobe-immobilized carrier, wherein at least one first probe according toclaim 1 is arranged on a solid-phase carrier.
 3. A probe-immobilizedcarrier according to claim 2, wherein the probe-immobilized carriercomprises at least one second probe having any one of the base sequencesof SEQ ID NOS. 35 to 94 as mentioned in the specification immobilized ata position spaced from the first probe.
 4. A kit for detecting a gene ofFlavobacterium (genus), comprising: a probe according to claim 1; and areagent for detecting a reaction between the probe and a target nucleicacid.
 5. A kit according to claim 4, wherein the reagent contains aprimer for amplifying the gene of Flavobacterium (genus), and the primerincludes: (3) an oligonucleotide having a base sequence of 5′gcggcaggcttaacacatgcaag 3′ (SEQ ID NO: 3); and (25) an oligonucleotidehaving a base sequence of 5′ atccagccgcaccttccggtac 3′ (SEQ ID NO: 25).6. A gene detection kit, comprising: a probe-immobilized carrieraccording to claim 3; and a reagent for detecting a reaction between theprobes and a target nucleic acid, wherein the reagent contains a primerincluding at least one oligonucleotide selected from the following items(1) to (21) and at least one oligonucleotide selected from the followingitems (22) to (28): (1) an oligonucleotide having a base sequence of 5′gcggcgtgcctaatacatgcaag 3′ (SEQ ID NO: 1); (2) an oligonucleotide havinga base sequence of 5′ gcggcaggcctaacacatgcaag 3′ (SEQ ID NO: 2); (3) anoligonucleotide having a base sequence of 5′ gcggcaggcttaacacatgcaag 3′(SEQ ID NO: 3); (4) an oligonucleotide having a base sequence of 5′gcggtaggcctaacacatgcaag 3′ (SEQ ID NO: 4); (5) an oligonucleotide havinga base sequence of 5′ gcggcgtgcttaacacatgcaag 3′ (SEQ ID NO: 5); (6) anoligonucleotide having a base sequence of 5′ gcgggatgccttacacatgcaag 3′(SEQ ID NO: 6); (7) an oligonucleotide having a base sequence of 5′gcggcatgccttacacatgcaag 3′ (SEQ ID NO: 7); (8) an oligonucleotide havinga base sequence of 5′ gcggcatgcttaacacatgcaag 3′ (SEQ ID NO: 8); (9) anoligonucleotide having a base sequence of 5′ gcggcgtgcttaatacatgcaag 3′(SEQ ID NO: 9); (10) an oligonucleotide having a base sequence of 5′gcggcaggcctaatacatgcaag 3′ (SEQ ID NO: 10); (11) an oligonucleotidehaving a base sequence of 5′ gcgggatgctttacacatgcaag 3′ (SEQ ID NO: 11);(12) an oligonucleotide having a base sequence of 5′gcggcgtgcctaacacatgcaag 3′ (SEQ ID NO: 12); (13) an oligonucleotidehaving a base sequence of 5′ gcggcgtgcataacacatgcaag 3′ (SEQ ID NO: 13);(14) an oligonucleotide having a base sequence of 5′gcggcatgcctaacacatgcaag 3′ (SEQ ID NO: 14); (15) an oligonucleotidehaving a base sequence of 5′ gcggcgcgcctaacacatgcaag 3′ (SEQ ID NO: 15);(16) an oligonucleotide having a base sequence of 5′gcggcgcgcttaacacatgcaag 3′ (SEQ ID NO: 16); (17) an oligonucleotidehaving a base sequence of 5′ gcgtcatgcctaacacatgcaag 3′ (SEQ ID NO: 17);(18) an oligonucleotide having a base sequence of 5′gcgataggcttaacacatgcaag 3′ (SEQ ID NO: 18); (19) an oligonucleotidehaving a base sequence of 5′ gcgacaggcttaacacatgcaag 3′ (SEQ ID NO: 19);(20) an oligonucleotide having a base sequence of 5′gctacaggcttaacacatgcaag 3′ (SEQ ID NO: 20); (21) an oligonucleotidehaving a base sequence of 5′ acagaatgcttaacacatgcaag 3′ (SEQ ID NO: 21);(22) an oligonucleotide having a base sequence of 5′atccagccgcaccttccgatac 3′ (SEQ ID NO: 22); (23) an oligonucleotidehaving a base sequence of 5′ atccaaccgcaggttcccctac 3′ (SEQ ID NO: 23);(24) an oligonucleotide having a base sequence of 5′atccagccgcaggttcccctac 3′ (SEQ ID NO: 24); (25) an oligonucleotidehaving a base sequence of 5′ atccagccgcaccttccggtac 3′ (SEQ ID NO: 25);(26) an oligonucleotide having a base sequence of 5′atccagcgccaggttcccctag 3′ (SEQ ID NO: 26); (27) an oligonucleotidehaving a base sequence of 5′ atccagccgcaggttctcctac 3′ (SEQ ID NO: 27);and (28) an oligonucleotide having a base sequence of 5′atccagccgcacgttcccgtac 3′ (SEQ ID NO: 28).
 7. A probe set for detectinga gene of infectious disease pathogenic bacterium, Flavobacterium(genus), including at least two probes selected from the following items(A) to (L): (A) a probe having a base sequence represented byGGAAGTAACTAGAATATGTAGTGTAGCGGTG (SEQ ID NO. 73); (B) a probe having abase sequence represented by GCCAGTGCAAACTGTGAGGAAGGT (SEQ ID NO. 74);(C) a probe having a base sequence represented byGGGTAGGGGTCCTGAGAGGGAGATC (SEQ ID NO. 75); (D) a probe having acomplementary sequence of the base sequence represented by SEQ ID NO.73; (E) a probe having a complementary sequence of the base sequencerepresented by SEQ ID NO. 74; (F) a probe having a complementarysequence of the base sequence represented by SEQ ID NO. 75; (G) a probehaving a modified sequence obtained by base deletion, substitution, oraddition on the base sequence represented by SEQ ID NO. 73 as far as itretains the function of a probe for detecting the gene of Flavobacterium(genus); (H) a probe having a modified sequence obtained by basedeletion, substitution, or addition on the base sequence represented bySEQ ID NO. 74 as far as it retains the function of a probe for detectingthe gene of Flavobacterium (genus); (I) a probe having a modifiedsequence obtained by base deletion, substitution, or addition on thebase sequence represented by SEQ ID NO. 75 as far as it retains thefunction of a probe for detecting the gene of Flavobacterium (genus);(J) a probe having a modified sequence obtained by base deletion,substitution, or addition on the complementary sequence of the basesequence represented by SEQ ID NO. 73 as far as it retains the functionof a probe for detecting the gene of Flavobacterium (genus); (K) a probehaving a modified sequence obtained by base deletion, substitution, oraddition on the complementary sequence of the base sequence representedby SEQ ID NO. 74 as far as it retains the function of a probe fordetecting the gene of Flavobacterium (genus); and (L) a probe having amodified sequence obtained by base deletion, substitution, or additionon the complementary sequence of the base sequence represented by SEQ IDNO. 75 as far as it retains the function of a probe for detecting thegene of Flavobacterium (genus).
 8. A probe-immobilized carrier accordingto claim 2, wherein the at least one first claim includes at least twofirst probes selected from the following items (A) to (L): (A) a probehaving a base sequence represented by GGAAGTAACTAGAATATGTAGTGTAGCGGTG(SEQ ID NO. 73); (B) a probe having a base sequence represented byGCCAGTGCAAACTGTGAGGAAGGT (SEQ ID NO. 74); (C) a probe having a basesequence represented by GGGTAGGGGTCCTGAGAGGGAGATC (SEQ ID NO. 75); (D) aprobe having a complementary sequence of the base sequence representedby SEQ ID NO. 73; (E) a probe having a complementary sequence of thebase sequence represented by SEQ ID NO. 74; (F) a probe having acomplementary sequence of the base sequence represented by SEQ ID NO.75; (G) a probe having a modified sequence obtained by base deletion,substitution, or addition on the base sequence represented by SEQ ID NO.73 as far as it retains the function of a probe for detecting the geneof Flavobacterium (genus); (H) a probe having a modified sequenceobtained by base deletion, substitution, or addition on the basesequence represented by SEQ ID NO. 74 as far as it retains the functionof a probe for detecting the gene of Flavobacterium (genus); (I) a probehaving a modified sequence obtained by base deletion, substitution, oraddition on the base sequence represented by SEQ ID NO. 75 as far as itretains the function of a probe for detecting the gene of Flavobacterium(genus); (J) a probe having a modified sequence obtained by basedeletion, substitution, or addition on the complementary sequence of thebase sequence represented by SEQ ID NO. 73 as far as it retains thefunction of a probe for detecting the gene of Flavobacterium (genus);(K) a probe having a modified sequence obtained by base deletion,substitution, or addition on the complementary sequence of the basesequence represented by SEQ ID NO. 74 as far as it retains the functionof a probe for detecting the gene of Flavobacterium (genus); and (L) aprobe having a modified sequence obtained by base deletion,substitution, or addition on the complementary sequence of the basesequence represented by SEQ ID NO. 75 as far as it retains the functionof a probe for detecting the gene of Flavobacterium (genus), and whereinthe at least two first probes are arranged on a solid-phase carrier atintervals from each other.
 9. A probe-immobilized carrier according toclaim 8, wherein the probe-immobilized carrier comprises at least onesecond probe having any one of the base sequences of SEQ ID NOS. 35 to94 as mentioned in the specification immobilized at a position spacedfrom the at least two first probes.
 10. A method of detecting a gene ofFlavobacterium (genus) in an analyte by using a probe-immobilizedcarrier, comprising the steps of: (i) reacting the analyte with aprobe-immobilized carrier according to claim 2; and (ii) detecting thepresence or absence of a reaction of the probe on the probe-immobilizedcarrier with a nucleic acid in the analyte, or detecting the strength ofa hybridization reaction of the probe on the probe-immobilized carrierwith a nucleic acid in the analyte.
 11. A method according to claim 10,further comprising the step of carrying out PCR amplification of thetarget nucleic acid in the analyte by using a primer including thefollowing oligonucleotides: (3) an oligonucleotide having a basesequence of 5′ gcggcaggcttaacacatgcaag 3′ (SEQ ID NO: 3); and (25) anoligonucleotide having a base sequence of 5′ atccagccgcaccttccggtac 3′(SEQ ID NO: 25).